David H. Oi

ANT BEHAVIOR AND MICROBIAL PATHOGENS (HYMENOPTERA: FORMICIDAE)

The effectiveness of microbial controls for pest ants can be reduced by ant behaviors. Introductions of pathogens, including nematodes, into ant nests result in behavioral responses by ants that affect infection rates to ants exposed to inocula, affect the dissemination of inocula among nestmates, and affect the dispersal of inocula outside the nest. These behaviors include grooming, secretion of antibiotics, nest hygiene, avoidance, and dispersal. Ant behaviors must be considered in developing microbial control agents. Approaches to overcoming the behavioral responses of the red imported fire ant to the entomopathogen Beauveria bassiana (Balsamo) Vuillemin are discussed.

Impact of Thelohania solenopsae (Microsporidia: Thelohaniidae) on Polygyne Colonies of Red Imported Fire Ants (Hymenoptera: Formicidae)

Abstract Three studies were conducted to assess the effects of the entomopathogen Thelohania solenopsae on polygynous, red imported fire ant, Solenopsis invicta Buren, colonies. A total of 57 of 122 queens (46.7%) from nine, field-collected, polygyne, S. invicta colonies, was infected with T. solenopsae. Infection rate of queens for each colony ranged from 25 to 75%. Laboratory colonies of polygyne S. invicta, with three to 12 queens, were inoculated and infected with T. solenopsae. Brood levels in all infected colonies declined to 0 after 26–52 wk. Brood did not reappear in any of the colonies after 3–11 wk, even though in two of the eight infected colonies, five fertile queens that were uninfected were recovered. Thus, polygyne, S. invicta colonies infected with T. solenopsae, which were confined and isolated under laboratory conditions, did not recover. Field plots that contained polygynous S. invicta colonies, which were infected with T. solenopsae, were monitored over a 2-yr period. Infection rates increased during the study and reached a maximum of 93%. Fire ant nest density and colony sizes fluctuated over time, with maximum reduction of 63% per plot. In general, fire ant reductions were attributed to smaller colony sizes. T. solenopsae infections in polygynous S. invicta can result in a slow colony decline and death. Under field conditions, the prolonged colony death may mask the impact of T. solenopsae by allowing for concurrent reinfestations.

Detection of Thelohania solenopsae (Microsporidia: Thelohaniidae) in Solenopsis invicta (Hymenoptera: Formicidae) by multiplex PCR

Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.

Fire Ant Control with Entomopathogens in the USA

Fire ants are stinging invasive ants from South America that infest over 129.5 million hectares in the southern United States, where eradication is no longer considered possible. The biological control of fire ants, especially by pathogens, is viewed by some as the only sustainable tactic for suppression. Microscopic-based surveys conducted in South America during the 1970s and 1980s led to the discovery of fungi and microsporidia infecting fire ants. Three of these microorganisms have been studied extensively: Beauveria bassiana 447, Thelohania solenopsae, and Vairimorpha invictae. B. bassiana 447 causes fire ant mortality but infections do not spread to queens and intercolony transmission was not evident. T. solenopsae has been found in the US and has been shown to spread naturally and debilitate colonies. Colony decline has also been associated with V. invictae, which is currently being evaluated for host specificity and possible release in the US. Through the use of molecular techniques, viruses infecting fire ant in the US have been discovered and characterized. Solenopsis invicta virus-1 can be transmitted easily to uninfected colonies and colony death often results. This virus apparently causes persistent, asymptomatic infections that actively replicate when the host is stressed. Research on fire ant-specific microsporidia and viruses, as well as other fire ant entomopathogens, is summarized to illustrate the efforts that have been undertaken to understand the biology of these pathogens and to facilitate their utilization in biological control of fire ants.

Yellow-head disease caused by a newly discovered Mattesia sp. in populations of the red imported fire ant, Solenopsis invicta

Recent surveys conducted in Florida, USA to determine the presence of Thelohania solenopsae revealed a new protozoan in S. invicta populations. The pathogen was first observed in workers collected from southwest Alachua Co., Florida. Large workers and female alates exhibited atypical yellow-orange colouration in the head and sometimes in the thorax, hence the designation yellow-head disease. The abdomen of the infected ants is normally as dark as those of older ants. Examination of the body contents of the ants showed many spindle-shaped, paired oocysts in the cuticle in all body regions and inside different body parts including the head and appendages. Further morphological examination of the oocysts identified the pathogen as belonging to the genus Mattesia. Notes on the distribution and pathogenesis of the protozoa are presented.

Loss of microbial (pathogen) infections associated with recent invasions of the red imported fire ant Solenopsis invicta

Loss of natural enemies during colonization is a prominent hypothesis explaining enhanced performance of invasive species in introduced areas. Numerous studies have tested this enemy release hypothesis in a wide range of taxa but few studies have focused on invasive ants. We conducted extensive surveys for the presence of six microbes in recently established populations (California, Australia, New Zealand, Taiwan, and China) of the invasive fire ant Solenopsis invicta. These microbes include Wolbachia, two microsporidia (Kneallhaziasolenopsae and Vairimorpha invictae) and three RNA viruses (SINV-1, -2 and -3), all of which previously have been reported in native South American populations of S. invicta. These surveys showed that the total number of enemy species is lower in the recently invaded areas compared with both South American and US populations. Only two microbes were found in any of these recently invaded areas: SINV-1 was detected in all surveyed populations except Australia and New Zealand, and SINV-2 was detected in California and Taiwan only. These results support the general prediction that invasive species lose many of their natural enemies during invasion. Further, the conspicuous absence of some of these microbes in these areas may result from strong selection against founders due to fitness costs associated with harboring detrimental infections rather than the alternative hypothesis that they simply were absent among the original founders. While the successful invasion of S. invicta in these recently invaded areas may be explained partly by the absence of natural enemies, other factors likely have been important as well.

Metatranscriptomics and Pyrosequencing Facilitate Discovery of Potential Viral Natural Enemies of the Invasive Caribbean Crazy Ant, Nylanderia pubens

Background Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens. Methodology and Principal Findings Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens. Conclusions Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest.

Speed of Efficacy and Delayed Toxicity Characteristics of Fast-Acting Fire Ant (Hymenoptera: Formicidae) Baits

Efficacy and speed of action of fire ant (Hymenoptera: Formicidae) baits that claim fast control of colonies were compared with a standard bait. More than 85% of red imported fire ant, Solenopsis invicta Buren, laboratory colonies provided bait containing the active ingredient indoxacarb died within 3 d, and all colonies were dead in 6 d. Standard bait containing hydramethylnon resulted in death of 60% of the colonies in 9 d. Bait containing spinosad did not cause colony death. Under field conditions, one-half of the areas treated with the indoxacarb bait did not have any active fire ant nests within 3 d, whereas 11 d was needed to reach the same level of control with the hydramethylnon bait. Spinosad had a maximum of 17% of the treated areas without nests after 3 d. The delay in death of S. invicta adults treated in the laboratory with the indoxacarb and spinosad baits was shorter than the standard hydramethylnon bait, which had mortality similar to the traditional delayed toxicity criterion of < 15% mortality after 24 h and > 89% mortality over the test period. Indoxacarb caused mortality of 57% at 24 h and 100% at 48 h; however, visual symptoms of toxicity were not readily observed for at least 8 h before the abrupt increase in death. Spinosad caused 96% mortality by 24 h, and initial mortality became apparent at 4 h. Time required for death of 15% of a treated population (LT15) of spinosad, indoxacarb, and hydramethylnon was 3, 9, and 16 h, respectively. Delayed toxicity characteristics of the fast-acting indoxacarb bait may be useful for the development of other fast-acting ant baits.

Field host range of the fire ant pathogens Thelohania solenopsae (Microsporida: Thelohaniidae) and Vairimorpha invictae (Microsporida: Burenellidae) in South America

We studied the field host specificity of the microsporidia Thelohania solenopsae and Vairimorpha invictae and their prevalence in the imported fire ants, Solenopsis invicta and S. richteri. Terrestrial ants were sampled by using bait traps and/or nest sampling at preselected sites in Argentina and Brazil. The sampling included the genera Solenopsis, Pheidole, Camponotus, Crematogaster, Linepithema, Brachymyrmex, Paratrechina, Dorymyrmex, and Wasmannia. The samples were examined under a phase-contrast microscope for the presence of microsporidian infections. The bait trap sampling revealed that: (1) T. solenopsae infected only S. richteri, S. invicta, and Solenopsis sp. at 6–67% of the sites and in 1.5–29% of the traps; (2) V. invictae infected only S. invicta at 6% of the sites and in 3% of the samples. The nest sampling revealed that: (1) T. solenopsae infected S. invicta, S. richteri, and S. macdonaghi, at 41–67% of the sites and in 11–58% of the colonies; (2) V. invictae infected the same species at 15–50% of the sites and in 2–26% of the colonies. We detected T. solenopsae and V. invictae in equal percentages of S. invicta sites (41%); however, the percentage of colonies infected with V. invictae was 20% and with T. solenopsae only 11%. At S. richteri sites, in contrast, T. solenopsae occurred at 46% of the sites and 15% of the colonies and V. invictae occurred at only 15% of the sites and 2% of the colonies. In S. macdonaghi, T. solenopsae was detected at 67% of the sites and 58% of the colonies, and V. invictae was detected at 50% of the sites and 26% of the colonies. This is the first report of V. invictae infecting S. macdonaghi. The proportion of S. richteri and S. invicta infected with T. solenopsae was similar. In contrast, the proportion of S. invicta infected with V. invictae was higher than S. richteri. We conclude that the microsporidia, T. solenopsae and V. invictae, show a very high specificity for Solenopsis ants in the field. It appears that T. solenopsae infects S. invicta and S. richteri equally but V. invictae may be more adapted to infect S. invicta. Published by Elsevier Science (USA).

Prevalence of Thelohania solenopsae (Microsporidia: Thelohaniidae) Infection in Monogyne and Polygyne Red Imported Fire Ants (Hymenoptera: Formicidae)

Abstract We determined the prevalence of natural field infections of the fire ant pathogen Thelohania solenopsae Knell, Allen, and Hazard in the monogyne and polygyne social forms of the red imported fire ant, Solenopsis invicta Buren, in three pastures in Florida. Social form was determined by examining the genotype of ants at the Gp-9 locus. The monogyne form contains a single fertilized queen per colony, and colony members have a genotype of Gp-9BB. In contrast, the polygyne form contains many fertilized queens per colony, and all three genotypes of Gp-9BB, Gp-9Bb, or Gp-9bb can be present within a colony. Among the study sites, ratios of monogyne:polygyne colonies ranged from 3:55–28:22, and infections rates were 42–78% when both social forms were included in the samples from each site. However, T. solenopsae infections were restricted to colonies of the polygynous social form. While T. solenopsae was only detected in polygynous colonies in the field, T. solenopsae infections were found in ants with the monogyne genotype. Ants from four colonies reared from field-collected, newly mated queens that were naturally infected with T. solenopsae were found to exhibit this genotype. T. solenopsae also was detected in individual alate female reproductives possessing the monogyne genotype, which were collected from polygynous colonies. Polygynous colonies can contain individual ants that possess either the polygyne or monogyne genotype. Thus, T. solenopsae infections can occur in fire ants with genotypes of either social form. Because making genotypic determinations of S. invicta social forms may be impractical in the field, we compared visual and genotypic determinations of polygyny and monogyny. Visual determinations, based mainly on the relative preponderance of major workers, corresponded to 85% of the genotypic determinations.